Device for measuring an analyte

ABSTRACT

A novel method for detecting the concentration of a metabolite in a fluid sample is provided. Devices for the detection of the analyte are also provided. In particular, a device for determining the concentration of 11-dehydro thromboxane in a urine sample and comparing it to a set of standardized quartile concentrations is provided. A concentration of urinary 11-dehydro thromboxane that falls within the fourth quartile is indicative of a greatly increased risk of a recurrent cardiovascular event.

RELATED APPLICATIONS

This application is a continuation-in-part of application No.PCT/CA03/00422 filed on Mar. 24, 2003 which claims priority under 35 USC§ 119(e) to U.S. Provisional Application No. 60/367,883 filed Mar. 24,2002, the entire contents of which are hereby incorporated by referencein their entirety.

FIELD OF THE INVENTION

The present invention relates to the rapid detection of an analyte in abiological sample. Particularly the invention relates to methods anddevices for the measurement of suppression of thromboxane generation inresponse to treatment with aspirin.

BACKGROUND OF THE INVENTION

Cardiovascular disease ranks as a leading cause of mortality andmorbidity and represents a significant drain on health resources in manycountries.

It is well established that aspirin therapy reduces the risk of a strokeand a first heart attack in healthy individuals, and subsequent heartattacks, strokes, or cardiovascular death in patients with establishedcardiovascular disease. For example, U.S. Pat. No. 5,240,917 relates tothe percutaneous administration of aspirin as an antithrombotic agent.

Studies have shown that aspirin reduces the risk of cardiovascularevents by as much as 25% in patients with arterial vascular disease.Most heart attacks and strokes are caused by blood clots in the heart orbrain arteries that form on top of cracked atherosclerotic plaques.These blood clots are predominantly composed of clumped platelets.Aspirin works to prevent blood clot formation at these sites by reducingthe ability of the platelets to clump together and form plateletaggregates. Aspirin, also known as acetylsalicylic acid, reducesplatelet reactivity because its acetyl group acetylates a keyintra-platelet enzyme known as cyclo-oxygenase. Once acetylated,cyclo-oxygenase cannot work to generate thromboxane A2, a substancereleased from the platelets that serves to activate other platelets andinduce them to clump together in aggregates. In order for aspirin towork, therefore, it must reduce thromboxane A2 levels.

Thromboxane A2 has a very short half-life, and is rapidly converted to astable metabolite called thromboxane B2. Although thromboxane B2 can bemeasured in blood, the tests can be problematic because platelets can beactivated during the collection process. Once activated, the plateletswill release thromboxanes that can interfere with the assay. It istherefore preferable to measure thromboxane B2 in the urine.

Even though platelets are an important part of blood clots, rapidtechnology to measure and predict platelet physiology is lacking. Someaccepted laboratory methods include:

-   -   i) Bleeding Time, a test which is qualitative, not quantitative;    -   ii) Platelet Aggregometry. This test measures the clumping of        platelets in response to various stimuli. The test is arduous,        time-consuming, and expensive and is not specific for the        effects of aspirin on platelet activation; and    -   iii) Tests of platelet activation using fluorescent cell sorting        techniques. This test can only be done on freshly collected        blood and uses size separation to separate platelets from other        blood cells and fluorescently-tagged antibodies to identify        activated platelets. This test is cumbersome and does not        provide aspirin-specific information.

The present invention provides a novel method for assessing plateletfunction and correlating a readout of that function with the risk of acardiovascular event.

Aspirin is effective for patients with heart attacks, strokes orperipheral arterial disease or those at risk of these disorders. Aspirinhas also been shown to be effective in reducing the incidence ofpregnancy-induced hypertension and pre-eclamptic toxicity in women atrisk. A role for aspirin in reducing the risk of fatal colon cancer hasalso been suggested and aspirin may be useful in the treatment ofpatients with antiphospholipid antibodies, including the lupusanticoagulant. Thus, determining the effectiveness of aspirin treatmentin many conditions is an important prognostic factor and may helpphysicians recommend the most appropriate therapeutic course.

While aspirin is effective in many individuals, approximately 10 to 20%of patients with arterial thrombosis who are treated with aspirin have arecurrent vascular event during long-term follow-up. The failure ofthese patients to derive a beneficial effect from aspirin is termed“aspirin resistance”. There are several possible explanations foraspirin resistance but, whatever the underlying cause, the result is thesame. It would obviously be beneficial to be able to identify thosepatients who are aspirin resistant in order to help physicians determinethe advisability of altering the aspirin dose or administeringalternative or additional anti-platelet therapies. A need thereforeexists for a simple method to accurately determine the response toaspirin and predict the likelihood of onset of a cardiovascular event orother medical condition that would benefit from lowering ofthromboxane-A2 levels.

SUMMARY OF THE INVENTION

To be able to identify those people at particular risk of having arecurrent vascular event, so that they can be appropriately treatedbefore a heart attack or stroke occurs, would be of great clinicalimportance. Former attempts to develop predictive assays, particularlythose utilizing blood, have had mixed results. Thus, it is an object ofone aspect of the present invention to provide a rapid, non-invasive,reproducible method for determining aspirin resistance. The presentinvention demonstrates for the first time an association between aspirinresistance, defined as failure of suppression of thromboxane generation,and cardiovascular risk. Determination of the degree of resistance toaspirin is used to predict the risk of a cardiovascular event or othercondition that would benefit from lowering thromboxane A2 levels.

The present invention is based on the observation that urinarythromboxane A2 metabolite levels in patients are a surprisingly accuratepredictor of recurrent cardiovascular mortality. Thus, determination ofmetabolite levels in patients may serve to identify those patients atparticular risk of developing cardiac ischemia or stroke.

In one aspect of the invention, a method for assessing aspirinresistance in a patient is provided. The method comprises determiningthe concentration of a metabolite of thromboxane A2 in a sample of bodyfluid from the patient. The method preferably further comprises the stepof comparing the concentration of metabolite in the sample to apredetermined set of concentration quartiles to determine within whichquartile the sample falls and determining aspirin resistance based onthe quartile of the sample. A concentration of metabolite within thesecond, third or fourth quartile is indicative of an increased risk of acardiovascular event.

In another aspect, a method for assessing the risk of a cardiovascularevent in a patient is provided. The method comprises determining thelevel of thromboxane B2 or another thromboxane A2 metabolite in a bodyfluid, preferably urine. In a preferred embodiment, the method comprisesan immunoassay in which a body fluid sample from the patient iscontacted with an antibody that specifically binds to a metabolite ofthromboxane-A2. The formation of immune complexes is then detected todetermine the level of antigen in the sample and the sample level thusobtained is compared to control levels to determine a relative riskfactor.

In another aspect, there is provided a method of screening a patient forrisk of having a cardiovascular event which comprises contacting a bodyfluid sample from the patient with an antibody which specifically bindsto a thromboxane-A2 metabolite, determining the degree of immune complexformation by immunoassay, and assessing the patient's risk of acardiovascular event upon the basis of immune complex formation.

In a preferred embodiment, the patient has arterial vascular disease andthe method is used to predict the risk of a recurrent vascular event.

In a further preferred embodiment, the metabolite that is measured isthromboxane-B2 metabolite, preferably 11-dehydro thromboxane B2.

In a further aspect, a urine level of this metabolite of greater than 15ng/mmol creatinine is indicative of risk of a cardiovascular event, morepreferably a urine level greater than 21.9 ng/mmol creatinine isindicative of risk of a cardiovascular event and most preferably a urinelevel greater than 33.8 ng/mmol creatinine is indicative of risk of acardiovascular event.

The present invention also provides a kit for assessing aspirinresistance. The kit typically comprises (a) an antibody thatspecifically binds to a thromboxane A2 metabolite, and (b) a labeledsample of the metabolite.

In another aspect of the invention, a device for detecting an analyte ina test sample obtained from a mammal is provided. The device comprisesan immobilized moiety that specifically binds to the analyte and meansfor visually determining the level of the analyte. The moiety thatspecifically binds the analyte is preferably an antibody, an antibodyfragment, a single chain antibody or an antigen-binding domain of anantibody. The binding moiety is immobilized on a solid support selectedfrom the group consisting of glass, polystyrene, nylon, celluloseacetate, nitrocellulose and other polymers. The device may be in theformat of a dipstick. In a preferred embodiment, the analyte is11-dehydro thromboxane B2.

In yet another aspect of the invention a method of predicting increasedrisk of an increased risk for a recurrent cardiovascular event isprovided. The method comprises:

-   -   a) measuring the concentration of 11-dehydro thromboxane B2 in a        test urine sample;    -   b) comparing the concentration of the test sample to the        quartile concentration of a series of reference samples;    -   c) determining which quartile concentration the test sample        falls within; and    -   d) predicting the risk based on the corresponding quartile        concentration.

In a particularly preferred embodiment, an immunoassay device fordetecting the presence of an analyte is provided. The device comprises astrip that comprises a reagent that specifically binds to the analyte tobe tested. The reagent is preferably distributed in patches to detectdifferent amounts of the analyte.

In another embodiment, an immunoassay device is provided which comprisestwo strips that are reversibly attached. One strip comprises anabsorbent material capable of absorbing a predetermined volume of urineor other bodily fluid and the second strip comprises patches havingdifferent amounts of a moiety that specifically binds the analytes to betested.

In yet another embodiment, an immunoassay device is provided comprisinga first strip having an antibody moiety which specifically binds theanalyte to be determined and a second strip containing at least onestandardized concentration of the analyte to be determined, wherein uponaddition of a test sample, analyte in the test sample competes withanalyte on the second strip for binding by the antibody moiety.

The present invention provides a device for detecting an analyte in atest sample obtained from a mammal. The device comprises at least oneassay strip having a plurality of patch regions, each patch regioncontaining a unique, predetermined amount of a recognition moleculespecific for the analyte. Interaction between the analyte and therecognition molecule provides a dose dependent colorimetric readout onthe patch regions. Preferably, the recognition molecule is selected fromthe group consisting of an antibody, an antibody fragment, a singlechain antibody, an antigen binding domain of an antibody, a ligand or areceptor. The analyte is preferably 11-dihydro thromboxane B2.

In another embodiment, the device comprises a first assay stripcomprising an absorbent material capable of absorbing a predeterminedamount of fluid and a second non-absorbent assay strip comprising aspecific recognition molecule. The presence of an analyte in theabsorbed fluid can be detected by allowing the first strip to interfacewith the second strip and determining the amount of recognition moleculebound to the analyte. Upon interaction of the first and second strip,the recognition molecule migrates from the second strip to the firststrip and the amount of antibody remaining on the second strip isindicative of the concentration of analyte in the sample. Therecognition molecule is preferably an antibody and the analyte is athromboxane metabolite.

In yet another embodiment a device for the detection of an analyte isprovided. The device comprises a first strip of absorbent materialhaving a recognition molecule dispersed therein and a secondnon-absorbent strip have predetermined amounts of an analyte standardimmobilized thereon, wherein upon immersion of the device in a samplefluid the recognition molecules are mobilized to competitively bind tothe analyte on the second strip or the analyte in the sample fluidwhereby the amount of recognition molecule bound to the second strip isinversely proportional to the concentration of analyte in the samplefluid.

An immunoassay system for the detection of thromboxane B2 is providedwhich comprises:

-   -   a) a first strip having an antibody specific for thromboxane B2,        and    -   b) a second strip impregnated with a labeled standard        concentration of thromboxane B2,    -   wherein upon exposure of the device to a test sample,        thromboxane B2 in the test sample competes with the impregnated        thrombaxane B2 for binding to the antibody specific for B2 and        the level of thromboxane B2 in the sample is inversely        proportional to the amount of labeled thromboxane B2 remaining        on the second strip.

The methods and devices of the present invention can proactivelyidentify patients who are relatively resistant to anti-thrombotic dosesof aspirin and who may benefit from higher doses of aspirin oradditional or alternative therapies that can either block thromboxaneproduction or activity or inhibit another pathway of plateletactivation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates graphically the relationship between 11-dehydrothromboxane B2 levels and risk of a cardiovascular event;

FIG. 2 illustrates one embodiment of a test device according to thepresent invention;

FIG. 3 illustrates the test device of FIG. 2 in association with asecond strip;

FIG. 4 illustrates a preferred embodiment of a test device of thepresent invention; and

FIG. 5 illustrates yet another embodiment of a test device.

DETAILED DESCRIPTION OF THE INVENTION

Survivors of acute myocardial infarction are at greatly increased riskfor subsequent fatal and non-fatal cardiovascular events. Thisheightened risk is influenced by many factors, such as age, co-morbiddiseases and response to treatment.

The term “cardiovascular event(s) as used herein refers to coronaryand/or cerebrovascular event(s) including primary myocardial infarction,secondary myocardial infarction, angina pectoris (including unstableangina), congestive heart failure, sudden cardiac death, cerebralinfarction, syncope, transient ischemic attack and the like.

While aspirin has been known to reduce thromboxane-A2 levels, thepresent invention provides a novel method for determining aspirinresistance. The degree of aspirin resistance can be used to predict theoccurrence of a cardiovascular event based on the surprising result thatthere is a correlation between the level of 11-dehydro thromboxane B2and the incidence of myocardial infarction, stroke and cardiovasculardeath.

The present invention provides a method for determining the risk of arecurrent cardiovascular event based on the level of thromboxane-A2produced in response to treatment with aspirin. The level ofthromboxane-A2 generation can be determined by measuring urinary levelsof metabolites of thromboxane-A2. A preferred stable metabolite ofthromboxane-A2 which can be measured is 11-dehydro thromboxane B2.

In responsive individuals, aspirin reduces levels of thromboxane A2 byirreversibly acetylating the enzyme, cyclo-oxygenase 1. However, asubpopulation of individuals does not exhibit this inhibition ofthromboxane generation in response to aspirin. The incompletesuppression of thromboxane generation with the usual dose (75 to 325mg/d) of aspirin is termed aspirin resistance. In patients withcardiovascular disease taking aspirin, those who are aspirin resistantare more likely to have a recurrence of a cardiovascular event. Thepresent invention provides a method for identifying those patients whoare aspirin resistant. In addition to the prediction of a cardiovascularevent, aspirin resistance may be an important factor in the selection ofa treatment for other conditions that would benefit from a lowering ofthromboxane levels.

The present invention provides a method of determining the progressionof a disease associated with resistance to aspirin. Individuals with ahigher aspirin resistance status tend to resist treatment with aspirinand tend to have a greater incidence of conditions associated withuninhibited thromboxane 2 levels.

The present invention provides a method for establishing quartiles ofthromboxane B2 levels and correlating those quartile levels with risk ofa cardiovascular event. Based on the range of levels found in patientstreated with aspirin, four quartiles were established. The firstquartile comprised levels less than 15.1 pg/mmol creatinine. The secondquartile comprises levels between 15.1 to 21.8 pg/mmol creatinine. Thethird quartile comprises levels between 21.9 and 33.8 pg/mmol creatinineand the fourth quartile comprises levels greater than 33.8 pg/mmol. Itis clearly apparent that these ranges are approximate and that in anystudy the quartile ranges may vary. The odds ratio for an incidence of acardiovascular event over a five year period are 1.0, 1.3, 1.4, and 1.8for the first to fourth quartiles, respectively. Thus, the risk ofhaving a cardiovascular event over a study period of approximately 5years is 80% greater for those in the fourth quartile as opposed tothose in the first quartile.

The method for assessing the risk of a cardiovascular event comprisesmeasuring thromboxane B2 levels in urine and determining which quartilethe level falls within. The association of a test level within aquartile range is indicative of the long-term relative risk ofmyocardial infarction, stroke and vascular death. Urinary levels of 11dehydro thromboxane B2 that are predictive of future cardiovascularevents are generally greater than 15 pg/mmol of creatinine. Urinary11-dehydro thromboxane levels that are predictive of cardiovascularevents are preferably in a range of about 15 to 100 pg/mmol creatinine,more preferably 21 to 100 pg/mmol creatinine and most preferably in arange of 30 to 100 pg/mmol creatinine.

FIG. 1 illustrates the association between quartiles of 11-dehydrothromboxane B2 levels and composites of myocardial infarction (MI),stroke, or cardiovascular (CV) death that was seen in an exemplarystudy. The study on which these results are based is discussed infurther detail in Example 1 below. The results indicate that if the testvalue falls within the first quartile, there is an absolute risk ofapproximately 10%. If the test value falls within the second quartile,there is an absolute risk of about 13% over 5 years. If the test valuefall within the third quartile, the absolute risk is about 14% and ifthe value falls within the fourth quartile, the risk is about 18%.

The present invention provides a method of predicting the occurrence ofa cardiovascular event in a patient, wherein a body fluid of the patientis subjected in vitro to determination of the levels therein of athromboxane-A2 metabolite or a fragment thereof.

The invention also provides a method of screening patients, for risk ofhaving a cardiovascular event or other condition which would benefitfrom the reduction of thromboxane A2 levels, wherein a body fluid of thepatient is subjected in vitro to determination of the levels therein ofa thromboxane A2 metabolite or a fragment thereof and an assessment ofthe patient's risk is made upon the basis of those levels.

Such screening may be positive i.e. to identify those patients at riskand consequently in need of alternative treatment or negative toeliminate those patients who are not at significant risk from extensivefollow-up.

Various methods can be used to measure the level of thromboxane-A2metabolites in a sample of biological fluid. The level can be measuredusing a method selected from the group consisting of chromatography,immunoassay, spectroscopy and other quantitative methods known to thoseskilled in the art. The chromatographic method is preferably highperformance liquid chromatography (HPLC) or gas chromatography (GC). Thespectroscopic method is preferably selected from the group consisting ofultraviolet spectroscopy, infrared spectroscopy and nuclear magneticresonance spectroscopy.

Various types of immunoassays can be used. For example, a “sandwich”assay in which the metabolite is sandwiched between a capture antibodyimmobilized on a solid support and a detecting labeled can be used todetermine the amount of bound labeled antigen antibody complex.Alternatively a competition immunoassay may be used in which an antibodyis bound to a support which is contacted with an unknown quantity ofsample metabolite and labeled antigen of the same type. The amount oflabeled antigen bound to the support is indicative of the amount ofantigen in the sample.

The term “antibody” is used herein to refer to a monoclonal orpolyclonal antibody or an antibody fragment having specific bindingaffinity. The term “antibody fragment” refers to a portion of anantibody, such as an antigen binding domain, a hypervariable domain ofeither the heavy or light chain and the term also includes single chainantibodies.

Some examples of solid supports that can be used in the presentinvention include plates, tubes, polystyrene beads, nylon,nitrocellulose, cellulose acetate, glass fibers and other types ofporous polymers.

The methods of the present invention can be performed using immunoassaykits. The kits may be dip-stick, flow-through or migratory in design aswell as other formats known to those skilled in the art.

The immunoassay determination of thromboxane A2 metabolites can beperformed using monoclonal or polyclonal antibodies, which may be raisedusing techniques conventional in the art. For example, antibodies may bemade by injecting a host animal, e.g. a mouse or rabbit, with theantigen. The antigen may be conjugated with an immunogenic protein suchas PPD, a protein derivative of tuberculin, Keyhole Limpet Haemocyanin,BSA etc., to provide either a serum containing polyclonal antibodies orspleen cells for fusion to provide hybridomas or immortalised celllines. Other standard methods may also be used.

In a preferred embodiment, the immunoassay comprises the use of anantibody in immobilised form, e.g. on microtitre plates, membranes orbeads, to trap the antigen. In a sandwich assay, the bound antigen maybe labeled using additional soluble antibody, which may be monoclonal orpolyclonal and which may either carry a label or, more conveniently, mayitself be labeled subsequently by reaction with a secondary antibodycarrying a label.

Suitable labels include radionucleides, fluorophores, chemiluminescentlabels, bioluminescent labels, enzymes, for example as used in ELISAsystems, dyes or particles such as colloidal gold. The assay fordetermining the concentration can be in the format of an RIA, ELISA,immunofluorometric assay or immunoradiometric assay.

Alternatively, a competitive binding assay may be used, wherein a knownquantity of labeled metabolite is added to the analyte solution andcontacted with a limited quantity of the immobilised monoclonalantibody, whereby the amount of labeled antigen which is immobilised isinversely proportional to the amount of target antigen present in theanalyte.

In one aspect of the invention, a method for determining aspirinresistance is provided. The method comprises:

-   -   a) contacting a body fluid with an antibody reactive with        11-dehydro thromboxane for a time and under conditions        sufficient to form an antigen-antibody complex and detecting the        antigen-antibody complex formed;    -   b) quantitating the amount of complex formed in step a); and    -   c) comparing the amount of complex quantitated in step b) with        standard concentrations, wherein an elevated level of 11-dehydro        thromboxane correlates with aspirin resistance.

In a preferred embodiment, the method further comprises quantitating thedegree of aspirin resistance to predict the risk of a cardiovascularevent.

In another aspect of the invention, the components needed to perform theimmunoassay are supplied in kit form. Such a kit would comprise:

-   -   (a) an antibody capable of binding to 11-dehydro thromboxane A2,        the antibody in an immobilised form;    -   (b) a control preparation of 11 dehydro thromboxane B2; and    -   (c) a labelled secondary antibody specific for 11 dehydro        thromboxane B2.

In a preferred embodiment of the method of the invention, a quantitativedetermination of 11 dehydro thromboxane B2 levels in urine may beobtained, wherein a level greater than 15 ng/mmol creatinine isindicative of an increased risk of heart failure.

The body fluid on which the determination is performed may be any bodyfluid in which 11 dehydro thromboxane may be located. It is preferablyurine. In some cases it may be convenient to extract the peptide, orotherwise treat the sample prior to determination.

Because 11-dehydro thromboxane B2 is highly stable on storage, areliable prognosis may be obtained when the determination is performedon samples that have been stored for some time. This is advantageous inthat it facilitates assay reproducibility and it enables the assay to bedelayed post sample collection. Another advantage of the method of theinvention is that the 11 dehydro thromboxane B2 determination can beperformed with high specificity and sensitivity leading to an accurateand reliable prediction of recurrent cardiovascular events. Prior artmethods do not approach this level of accuracy and sensitivity.

The present invention provides a novel test kit or device for themeasurement of an analyte. The devices of the present invention may alsobe referred to as immunoassay systems. One preferred embodiment of adevice for the determination of analyte levels is shown in FIG. 2.Although thromboxane is referred to herein as an example of analyte thatcan be detected using the novel assay devices of the present invention,it is clearly apparent that the devices described herein can also beused to detect other analytes by using different reagents on the device.A test strip 10 is provided which is divided into test patches, 12, 14,16 and 18. Each patch has a reagent that reacts to the presence of11-dehydro thromboxane B2 or any other analyte to be detected. Eachreagent is applied so it will react with a predetermined concentrationof the analyte, e.g.11-dehydro thromboxane B2, in a given amount oftime. In a particularly preferred embodiment, each of the said patches,12, 14, 16 and 18 are adjusted to react to concentration of 11-dehydrothromboxane B2 in urine of a) less than 15.1, b) 15.1 to 21.8, c) 21.9to 33.8 and d) greater than 33.8 (expressed as ng/mmol creatinine),respectively. In other embodiments, the patches are adjusted to react tovarious concentrations of other analytes. The device is processed so adye or other calorimetric agent provides a readout of the level11-dehydro thromboxane B2 present in the urine. Various colorimetricagents can be used and the processing steps required for their detectionwill vary according to the agents. Methods for the detection ofcolorimetric agents in immunoassays are well known to those skilled inthe art. The test strip 10 may also include a patch 20 that changescolor when the patch has been in the urine or other fluid for anappropriate amount of time to obtain the desired reaction. The reagentsused for this patch 20 would react to known substances in the urine orother fluids. In some preferred embodiments the patches 12, 14, 16, 18also have printed on each of them the absolute and relative risk factorsassociated with the amount of 11-dehydro thromboxane B2 detected on thatparticular patch. This would allow the clinician to directly obtain therisk to the patient tested from the test strip itself. This would speeddiagnosis and avoid errors.

In a preferred embodiment shown in FIG. 3, the test strip 10 isreleasably attached to a second strip 22. The second strip 22 istypically made of an absorbent material and can absorb a predeterminedamount of urine. When the two strips are in contact with each other, themoieties on the two strips can interact to produce a signal. Typicallyan antibody, or other recognition molecule will react with an analytecontained in the urine absorbedonto the second strip. This test devicecan be used to detect the presence of and/or quantitate the amount of ananalyte in a sample. The sample may be urine, as mentioned above, or itmay be another fluid.

One preferred device according to the present invention is shown in FIG.4. This system includes two strips 30, 32 which are detachably joined sothat they can be immersed into the urine sample while bonded together,but can then be pulled or peeled apart at the appropriate time when theuser wishes to view the results of the reactions. In one preferredembodiment of the invention, strip 32 is made of an absorbent material,such as a porous micro filter polymer membrane or similar materialdesigned to absorb a fixed volume of urine when the two strips areimmersed in the urine sample. The other strip 30 comprises a polymermembrane or sheet made, for example, of nitrous cellulose or similarmaterial. This membrane is preferably impregnated with or coated with afixed concentration of a reporter molecule 34 such as a dye or enzymethat is linked to an antibody or other bioreceptor 36 specific for theanalyte to be detected, such as 11-dehydro thromboxane B2. Strip 30 ismade of a material that does not allow urine to be absorbed into it,preferably a hydrophobic material. The interface between strip 30 andstrip 32 allows the 11-dehydro thromboxane B2 contained in the urine instrip 32 to come in contact with the antibody specific for 11-dehydrothromboxane B2 contained in and/or on strip 30. Movement of labeledantibody between strips 30 and 32 may be facilitated by providing,between the antibody and strip 30, a separable physical bond 35 in amanner that retains adequate analyte recognition between the antibodyand the 11-dehydro thromboxane B2 associated with the strip. The bondmay be accomplished using IgG Fc region specific binding proteins suchas Protein A, Protein G, or secondary antibodies specific for the Fcregion of the primary antibody. This separable physical bond provides ameans of anchoring the antibody 36 to strip 30. After being immersed inthe urine, the two strips are incubated for a fixed time period and thenseparated. The antibody 36 is capable of recognizing the analyte 37present in the urine and will migrate to the strip 32. The presentinvention takes advantage of the affinity between the antibody and theanalyte, e.g. 11-dehydro thromboxane B2. In a preferred embodiment, theantibody remains on strip 30 if it does not react with 11-dehydrothromboxane B2 in the urine. If the antibody binds to 11-dehydrothromboxane B2 contained in the urine on strip 32 it will be removedfrom strip 30. A signalling moiety is associated with the antibody andthus, after processing of the strips, the amount of signal on strip 30is inversely proportional to the amount of 11-dehydro thromboxane B2contained in the urine when the two strips 30 and 32 are peeled apart.The strip optionally comprises at least two, preferably 4, referencepatches that allow comparison between the level of signal resulting fromthe reaction and the visual signal that corresponds to each of the fourlevels of 11-dehydro thromboxane B2 (less than 15.1, 15.1 to 21.8, 21.9to 33.7 and finally greater than 33.7 referred to above). This systemmay also indicate the corresponding risk levels that correspond to theamounts of 11-dehydro thromboxane B2 contained in the urine. Theconcentrations of antibody can be altered to provide optimal signal tonoise ratios. The device shown in FIG. 4 can also be modified for thedetection of other analytes by using different recognition molecules. Inaddition the test sample may comprise a fluid other than urine.

In another embodiment, shown in FIG. 5, strip 42 has a labeled antibody44 associated with it. Partner strip 46 may optionally comprise at leasttwo, preferably four, patches 48, 50 which contain increasing densitiesof synthetic or natural analyte (e.g. 11-dehydro thromboxane B₂) 56immobilized to the surface. When the device is immersed in the urinesample, the labeled antibody is mobilized to distribute itself betweenthe analyte in the sample 58 and the immobilized analyte 56 on strip 44.In other words, there is competitive binding between the analyte in theurine on one strip and the immobilized analyte on the partner strip. Thepresence of analyte in the urine sample blocks binding of the labeledantibody to the immobilized analyte on the patches in a dose-dependentmanner. The analyte is detected and quantified by the relative bindingto the patches of increasing density.

While preferred embodiments comprising two strips of material have beendescribed, it should be understood that these components could besegments of a larger belts or rollers. It is also clearly apparent thateither a positive (increase in signal intensity) or a negative (decreasein signal) readout can be used on either strip as an indicator of theamount of analyte in the test sample. The devices of the presentinvention can be used to detect the presence of virtually any analyte ina fluid sample as long as there is a recognition molecule specific forthat analyte. It is also apparent that while the devices of the presentinvention have been described with the example of determining quartileconcentrations of thromboxane, the strips do not have to be limited toquartile determinations. The reagents on the strips of the invention canbe selected for other types of concentration determinations. Forexample, the strips can be designed to detect only concentrations inexcess of a predetermined threshold amount or the absolute concentrationof an analyte can be determined. The single and dual strip devicesdescribed herein offer significant advantages over other immunoassaymethods. Only a small volume of test sample is required and a clinicallyrelevant readout can be quickly and easily obtained.

While the present invention has been described in conjunction withpreferred embodiments, the measurement of analyte or simultaneousmeasurement of two or more analytes (such as 11-dehydro thromboxane B₂and creatinine) can also be performed using existing rapid testingtechnologies such as, but not limited to biosensors or membrane baseddipstick, lateral flow or chromatographic strips.

While these embodiments have described a method for analyzing the amountof 11-dehydro thromboxane B₂ in a test sample, it should be understoodthat in other embodiments of this invention, other thromboxane A2metabolites can be measured as an indicator of aspirin resistance. It isapparent that for the detection of other metabolites, other antibodiesthat have an affinity for those metabolites will be substituted for thepurpose of analyzing the presence and amount of these other proteins.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Changes in form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein, such terms are intended in a descriptivesense and not for purposes of limitation.

As will be demonstrated in the examples to follow, the early detectionof aspirin resistance is an important indicator for improved long-termoverall survival and reduced mortality and morbidity due to majorcardiovascular events. In particular, 11-dehydro thromboxane levels ofgreater than 33.8 pg/mmol creatinine are associated with an 80% greaterrisk of a cardiovascular event than levels less than 15.2 pg/mmolcreatinine. By recognizing aspirin resistance and its implications,overall deaths can be reduced and congestive heart failure requiringhospitalization can be reduced. The detection of aspiring resistance isalso important for the development of an appropriate treatment strategyfor other condition which may benefit from a reduction thromboxane A2levels.

EXAMPLES

The examples are described for the purposes of illustration and are notintended to limit the scope of the invention.

Example 1

Study Design

The HOPE study¹ was an international, randomised, placebo-controlled,two-by-two factorial trial of ramipril and vitamin E for the secondaryprevention of cardiovascular disease. The institutional review committeeat each participating center approved the study and all subjects gaveinformed consent.

Patients

A total of 9,541 patients aged at least 55 years at the time ofrandomization who had a history of coronary artery disease, stroke,peripheral vascular disease, or diabetes plus at least one othercardiovascular risk factor were assigned to one of four treatments:ramipril titrated up to 10 mg daily, vitamin E 400 IU daily, both, orneither. The study commenced in December 1993 and was terminatedprematurely on Mar. 22, 1999 because of clear evidence of a benefit oframipril.

Urine Sample Collection

All study participants were asked to provide a first morning urinespecimen at the time of randomisation. Of the 9,541 patients in the HOPEstudy, 9,282 (97%) provided baseline urine samples. Samples (n=5529)from the 129 Canadian centres participating in this study were sent tothe central laboratory in Hamilton, Canada where they were stored at−80° C. until analysis. Only samples from Canadian centres were used forthe present study.

Follow-up and Ascertainment of Clinical Outcomes

All patients in the HOPE study were followed at one month, six months,and six monthly intervals thereafter until completion of the study. Ateach follow-up, clinical outcomes were recorded and medication use,including aspirin, was documented. The primary outcome was the compositeof myocardial infarction, stroke, and death from cardiovascular causes.

Selection of Cases and Controls

Of patients with available urine samples (n=5529), only those who weretaking aspirin at the time of commencement of the run-in phase (prior torandomisation), at randomisation (coinciding with the time of urinecollection), and at each follow-up visit, were eligible for inclusion.Aspirin-treated patients who provided an adequate baseline sample ofurine and had a confirmed myocardial infarction, stroke, orcardiovascular death after randomisation were defined as cases. Controlswere randomly selected from aspirin-treated patients who provided anadequate baseline urine sample but did not experience myocardialinfarction, stroke, or cardiovascular death after randomisation.Controls were matched according to gender and age (±5 years) in a 1:1ratio with cases.

Laboratory Analysis

For each case and control, urine collected and stored at baseline wasthawed and assayed for 11-dehydro thromboxane B₂ levels using acommercially available enzyme immunoassay (Cayman Chemical, Ann Arbor,Mich.) that has inter- and intra-assay coefficients of variation of12.1% and 10%, respectively. Assays were performed by laboratory staffblinded to patient status as case or control. In addition, case andcontrol specimens were assayed in random order, thereby reducing thepossibility of systematic bias.

Statistical Analysis

Means or proportions for baseline demographics and risk factors werecalculated for cases and controls. The significance of any differencebetween cases and controls was tested using Student's paired t-test formeans and McNemar chi square test for proportions, which takes intoaccount the matching between cases and controls. Because 11-dehydrothromboxane B₂ values are skewed, geometric means were calculated afterlog transformation of the raw data and the significance of anydifferences in geometric mean values between cases and controls wastested using Student's paired t-test. Median concentrations also werecalculated and levels in cases and controls were compared usingWilcoxon's rank-sum test.

Tests for trend were used to assess any association between increasingbaseline urinary 11-dehydro thromboxane B₂ concentrations and risk ofmyocardial infarction, stroke, or cardiovascular death after dividingthe samples into quartiles defined by the distribution of the completecohort. Adjusted estimates of the association between increasingbaseline urinary 11-dehydro thromboxane B₂ concentrations and risk ofmyocardial infarction, stroke, or cardiovascular death were obtainedusing conditional logistic regression modelling that accounted for thematching variables and controlled for the random treatment assignmentand baseline differences between cases and controls. A separatemultivariable regression model was used to examine the associationbetween baseline patient characteristics, including age, gender, heartrate, blood pressure, body mass index, past history of vascular disease,conventional vascular risk factors, lipid-lowering therapy,beta-blockers, diuretics, and randomised treatment allocation (ramiprilor vitamin E), and urinary 11-deydro thromboxane B₂ concentrations inthe urine.

All P-values are two-sided and confidence intervals are calculated atthe 95 percent level.

Baseline characteristics of cases and controls are shown in Table 1. Asexpected, patients in whom myocardial infarction, stroke, orcardiovascular death subsequently developed had a higher mean body massindex and baseline blood pressure and were more likely than those whoremained free of these events to be current smokers or have a history ofhypertension, diabetes, myocardial infarction, or peripheral vasculardisease. Cases also were more often treated with diuretics or calciumchannel blockers at baseline and less often treated with lipid-loweringdrugs or randomised to ramipril therapy. Because of the matching, theage and gender of cases and controls were similar.

Geometric mean and median urinary concentrations of 11-dehydrothromboxane B₂ at baseline were significantly higher among patients whosubsequently developed the composite outcome of myocardial infarction,stroke, or cardiovascular death compared with those who remained free ofthese events (Table 2). The difference between cases and controls wasgreatest in those who suffered a myocardial infarct (24.5 vs. 20.9ng/mmol creatinine, P=0.003) or died from a cardiovascular cause (25.6vs. 20.4 ng/mmol creatinine, P<0.001).

The adjusted odds for the composite outcome of myocardial infarction,stroke, or cardiovascular death increased with each increasing quartileof baseline urinary 11-dehydro thromboxane B₂ concentration (P for trendacross quartiles, 0.01), with patients in the highest quartile having arisk 1.8-fold higher than those in the lowest quartile (Odds Ratio [OR]1.8; 95 percent confidence interval [CI] 1.2-2.9, P=0.009) (FIG. 1). Asimilar association was seen with myocardial infarction (P for trendacross quartiles, 0.005) and cardiovascular death (P for trend acrossquartiles, 0.001) (Table 3). Results were similar with or withoutadjustment for baseline differences between cases and controls includingconventional vascular risk factors, co-interventions, and randomisedtreatment allocation.

To evaluate whether increased baseline urinary 11-dehydro thromboxane B₂concentrations were associated with early rather than latecardiovascular events separate analyses were performed in patients whoexperienced an event within the first 12 months of study entry and thosewhose event occurred more than 12 months after study entry. The adjustedodds for the composite outcome of myocardial infarction, stroke, orcardiovascular death that was associated with the highest quartile ofurinary 11-dehydro thromboxane B₂ as compared with the lowest quartilewas 2.9 (95% Cl: 0.9-9.1) for events occurring with the first 12 monthsand 1.7 (95% Cl: 1.0-2.7) for events occurring after the first 12months.

Using linear multivariable regression modeling, variables that werefound to be independently associated with baseline urinary 11-dehydrothromboxane B₂ concentrations in the urine were: female gender(P=0.004); body mass index (P=0.001), history of peripheral vasculardisease (P=0.01), current cigarette smoking (P=0.09), use of calciumchannel blockers (P=0.08), and randomisation to vitamin E (P=0.04).However, these variables combined were able to predict less than 5% ofthe variation in urinary 11-dehydro thromboxane B₂ concentrations(R-square 0.045).

These results indicate that urinary thromboxane B2 levels can be used anindicator of aspirin resistance and that aspirin resistance is avaluable predictor of the occurrence of a cardiovascular event.

TABLE 1 Baseline characteristics of study participants.* Cases ControlsCharacteristic† (n = 488) (n = 488) P-value Age - yr 67.3 ± 7.2   67.4 ±7.2   0.78 Female sex - no. (%)  77 (15.8)  77 (15.8) — Body mass index‡27.8 ± 4.1   26.9 ± 3.7   <0.001 Heart rate - beats/min  66.2 ± 10.3   65.6 ± 10.9   0.41 SBP - mm Hg 137.1 ± 20.6   133.5 ± 18.0   0.002DBP - mm Hg 76.6 ± 9.8   75.6 ± 9.4   0.08 History of coronary disease -no. (%) Any 469 (96.1) 464 (95.1) 0.54 MI 364 (74.6) 309 (63.4) <0.001Stable angina 355 (72.7) 336 (68.9) 0.19 Unstable angina 184 (37.7) 176(36.1) 0.65 CABG 176 (36.1) 154 (31.6) 0.15 PCI  87 (17.8) 104 (21.3)0.22 Stroke or TIA - no. (%)  59 (12.1) 40 (8.2) 0.06 Peripheralvascular 240 (49.2) 173 (35.5) <0.001 disease - no. (%) Hypertension -no. (%) 219 (44.9) 154 (31.6) <0.001 Diabetes - no. (%) 159 (32.6) 105(21.5) <0.001 Elevated total 279 (57.2) 310 (63.5) 0.38 cholesterol -no. (%) Current cigarette  81 (16.6)  57 (11.7) 0.03 smoking - no. (%)Medications - no. (%) Aspirin 488 (100)  488 (100)  — β-blocker 241(49.4) 235 (48.2) 0.76 Lipid-lowering agent 121 (24.8) 166 (34.0) 0.002Diuretics  73 (15.0) 34 (7.0) <0.001 Calcium channel 289 (59.2) 238(48.8) 0.002 blockers Ramipril 227 (46.5) 274 (56.1) 0.003 Vitamin E 246(50.4) 252 (51.6) 0.74 *Plus-minus values are mean ± SD. †CABG denotescoronary artery bypass graft surgery, CV cardiovascular, DBF diastolicblood pressure, MI myocardial infarction, PCI percutaneous coronaryintervention, SBP systolic blood pressure, TIA transient ischaemicattack. ‡The body-mass index is the weight in kilograms divided by thesquare of the height in meters.

TABLE 2 Baseline urinary concentrations of urinary 11-dehydrothromboxane B₂ in cases and contr ls. 11-dehydro thromboxane B₂concentration (ng/mmol creatinine) Outcome Cases Controls P-value MI,Stroke or CV death (n = 488) Geometric mean 24.5 21.5 0.01 Median 22.721.0 0.01 MI (n = 378) Geometric mean 24.5 20.9 0.003 Median 22.8 20.30.001 Stroke (n = 80) Geometric mean 25.0 27.4 0.47 Median 21.3 25.90.40 CV death (n = 244) Geometric mean 25.6 20.4 <0.001 Median 24.0 19.9<0.001 MI denotes myocardial infarction, CV cardiovascular.

TABLE 3 Adjusted odds* of future cardiovascular death, myocardialinfarction, and stroke according to baseline urinary concentrations of11-dehydro thromboxane B₂. Quartiles of 11-dehydro thromboxane B₂concentration (ng/mmol creatinine) Outcome† <15.1 15.1-21.821.9-33.7 >33.7 P for trend MI/Stroke/CV death (n = 488) Odds ratio (95CI) 1.0 1.3 (0.9-2.0) 1.4 (0.9-2.2)  1.8 (1.2-2.7) 0.01 P-value — 0.130.09  0.009 MI (n = 378) Odds ratio (95 CI) 1.0 1.3 (0.8-2.1) 1.5(1.0-2.5)  2.0 (1.2-3.4) 0.005 P-value — 0.26 0.07  0.006 Stroke (n =80) Odds ratio (95 CI) 1.0 2.5 (0.6-10.0) 0.6 (0.2-2.2)  0.6 (0.2-1.8)0.20 P-value — 0.18 0.45  0.34 CV death (n = 244) Odds ratio (95 CI) 1.02.0 (1.0-3.9) 2.5 (1.3-4.9)  3.5 (1.7-7.4) 0.001 P-value — 0.06 0.006<0.001 *Adjusted for baseline differences between cases and controls.†CI denotes confidence interval, MI myocardial infarction, CVcardiovascular.

REFERENCES

-   1. Eikelboom J. et al. Aspirin resistance and the risk of myocardial    infarction, stroke, or cardiovascular death in patients at high risk    for cardiovascular events. Circulation 2002; 105:1650.

1. An immunoassay kit for detecting an analyte in a test sample,comprising: i. a first strip including a test patch that contains apredetermined amount of an antibody specifically binding to the analyte,wherein the antibody is linked to a reporter molecule capable ofreleasing a signal, and ii. a second strip made of an absorbent materialfor absorbing the test sample the second strip being configured to beseparable from the first strip, wherein after contacting the first stripfor a fixed time period with the second strip absorbed with the testsample and then separating them, the antibody bound to the analyte movesfrom the first strip to the second strip and the antibody not bound tothe analyte remains on the first strip, the level of signal releasedfrom the reporter molecule linked to the antibody remaining on the firststrip being inversely proportional to the amount of the analyte in thetest sample.
 2. The immunoassay kit of claim 1, wherein the first stripfurther includes four reference patches containing four predeterminedamounts of the reporter molecule, the levels of signal releasedtherefrom corresponding to four determined concentrations of theanalyte.
 3. The immunoassay kit of claim 1, wherein the reportermolecule is a dye.
 4. The immunoassay kit of claim 1, wherein thereporter molecule is an enzyme.
 5. The immunoassay device according toclaim 1, wherein the antibody binds to thromboxane B2.
 6. Theimmunoassay kit according to claim 5, wherein the first strip furtherincludes four reference patches containing four predetermined amounts ofthe reporter molecule, the level of signal released therefromcorresponding to relative risks of a cardiovascular event.
 7. Theimmunoassay kit according to claim 6, wherein the levels of signalreleased from the four predetermined amounts of the reporter moleculecorrespond to the following four amounts of urinary 11-dehydrothromboxane B2: (i) less than 15.1 ng/mmol creatinine, (ii) 15.1-21.8ng/mmol creatinine, (iii) 21.9-33.8 ng/mmol creatinine, and (iv) higherthan 33.8 ng/mmol creatinine.
 8. The immunoassay kit according to claim6, wherein the reporter molecule is a dye.
 9. The immunoassay kitaccording to claim 6, wherein the reporter molecule is an enzyme.